Fluorogenic Method Research Articles

Determination of cysteine concentration by fluorescence increase: reaction of cysteine with a fluorogenic aldehyde.

A fluorogenic method for the determination of cysteine concentration has been developed.

Self-reporting PNA/DNA primers for PCR analysis.

We report a new fluorogenic method for sealed-tube PCR analysis using a quencher-labeled peptide nucleic acid (Q-PNA) probe. The Q-PNA hybridizes to a complementary tag sequence located at the 5' end of a 5' fluorophore-labeled oligonucleotide primer, quenching the primer's fluorescence. Incorporation of the primer into a doublestranded amplicon causes displacement of the Q-PNA such that the fluorescence of the sample is a direct indication of the amplicon concentration. The Q-PNA is able to quench multiple primers bearing distinct 5' fluorophores in a single reaction. We show realtime quantitative detection of a single-copy gene, K-ras, from human genomic DNA, as well as an endpoint multiplex assay for Chlamydia trachomatis and Neisseria gonorrhoeae targets. Because the Q-PNA may be used to quench any primer that contains the 5' tag sequence, it is possible to inexpensively adapt an existing primer set for use in a self-reporting fluorescent assay by including the tag sequence in one of the primers.

A fluorogenic allele-specific amplification method for DNA-based screening for inherited metabolic disorders

A fluorogenic allele-specific amplification method for DNA-based screening for inherited metabolic disorders

Evaluation of a rapid fluorogenic method for the detection ofEscherichia coliin dairy products

A rapid fluorogenic medium was evaluated for the detection of Escherichia coli in dairy products. The medium was capable of detecting Esch. coli after 7.5 h incubation at 41.5 degrees C. Samples of pasteurized milk (136), raw milk (63), soft cheese (60) and pasteurized cream (39) were examined with media based on 4-methylumbelliferyl-beta-D-glucuronide (MUG-7) and Violet red bile agar and there were no significant differences between the numbers of Esch. coli detected on the two media. MUG-7 medium had a specificity of 98.6% and the small number of organisms giving a false positive reaction were identified as Klebsiella pneumoniae. The incidence of false negative results was approximately 2%. MUG-7 medium was suitable for pour plate, spread plate and membrane filtration methods. Possible applications of the method are discussed.

A rapid fluorogenic method for the detection ofEscherichia coliby the production of β‐glucuronidase

A medium containing the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide was developed for the isolation and identification of Escherichia coli within 7.5 h and was based on the detection of beta-glucuronidase. Optimum conditions for the rapid development of fluorescent colonies were determined. The optimum temperature was 41.5 degrees C. Development of fluorescence was delayed when carbohydrates were incorporated into the medium. Water samples were used to evaluate the medium by surface plating and membrane filtration. The frequency of false-negative results was 6.1% and false-positives were 3.7% for freshwater samples. The false-positive organisms were identified as Klebsiella spp. and Shigella sonnei. The potential applications of the medium are discussed.

Modified most-probable-number technique for the specific determination of Escherichia coli from environmental samples using a fluorogenic method

A specific and senstive modification of the most-probable-number (MPN) technique by addition of 4-methylumbelliferyl-β-D-glucuronide (MUG) to both presumptive and confirmatory media was performed. The use of this modification allows the precise determination of Escherichia coli from marine samples (seawater, sediment and shellfish) within 7 days compared to 10–12 days required by using of the standard methodology. No false-positive isolates for fluorescence reaction have been observed, although one E. coli strain fluorescent-positive on agar was isolated from nonfluorescent tubes. Klebsiella pneumoniae was the species most frequently detected from tubes with gas and fluorescence production and typical morphology on mFC agar, but failed on nutrient-MUG agar. Using a unifactorial variance analysis of the mean, fluorescence in tube and on agar has been determined as the factor which allows the detection of E. coli presence in the three sample types with high accuracy. The incorporation of MUG to the selective broth in the confirmatory test has been shown to avoid false-positive results due to shellfish tissue β-glucuronidase activity. Specificity of the MUG-MPN method was high ( Se = 0.94 and Sp = 0.79). The results obtained using laboratory strains showed that although E. coli, Citrobacter freundii, Enterobacter cloacae and K. pneumoniae produce acid and gas in MacConkey-purple broth, β-glucuronidase activity in this selective broth is specific to E. coli.

Neutral proteases involved in the reinvasion of erythrocytes by Plasmodium merozoites.

Neutral proteases of Plasmodium sp erythrocytic stages were studied by means of a sensitive fluorogenic method and gelatin-SDS-PAGE. The substrates gluconoyl-Val-Leu-Gly-Lys(or Arg)-3-amido-9-ethylcarbazole were selectively hydrolyzed by an endopeptidase from rodent Plasmodium berghei (Pb) and Plasmodium chabaudi (Pc) and from human Plasmodium falciparum (Pf) parasites. These endopeptidases were purified from 100,000-g soluble schizont extract by high pressure liquid chromatography; they have a similar Mr of 68,000 in SDS-PAGE, and an optimal activity at pH 7.4. The Pb 68 and Pf 68 endopeptidases were localized in schizonts and also in merozoites as shown by indirect immunofluorescence on Pb merozoites and by the identification of the Pf 68 endopeptidase activity in free viable merozoites. The Pb 68 and Pf 68 endopeptidases belong to the class of cysteine proteases. Analysis by gelatin-SDS-PAGE of a Pb 68 endopeptidase-enriched fraction showed a reproducible 95,000 proteolytic band. The initial extracts showed a similar 95,000 proteolytic band, and also 2 other 90,000 and 85,000 major bands. During reinvasion experiments, it was possible to recover a 95,000 and a 40,000 protease band from supernates of cultures grown in a semidefined medium without serum. Hydrophilic peptide derivatives related to the substrate of Pf 68 endopeptidase are shown to be potential inhibitors of the Pf reinvasion process in vitro.

Activities and subcellular distributions of hepatic lipases in control subjects and in patients with alcoholic fatty liver.

Human hepatic lipase activities were studied in needle biopsy specimens by fluorogenic and radioisotopic assay methods. Using analytical subcellular fractionation by sucrose density gradient centrifugation a lysosomal acid lipase, with pH optimum of 4.0, and an endoplasmic reticulum alkaline lipase, with pH optimum of 8.5, were demonstrated with the fluorogenic assay. The apparent Km of the acid lipase was 17 mumol/l by the fluorogenic method and 23 mmol/l by the radioisotopic method. The values for alkaline lipase were 94 mumol/l and 1.4 mmol/l respectively. Assay of these activities in biopsies from 16 control subjects and 42 chronic alcoholics showed increasing activity with increasing hepatic fatty infiltration when the fluorogenic assay was used: no differences were demonstrated with radioisotopic assay. These results suggest that depressed lipolysis due to a relative deficiency of triglyceride lipase is not a causal factor in triglyceride accumulation in chronic alcoholic fatty liver.

Fluorescent staining of sialidases in polyacrylamide gel electrophoresis and ultrathin-layer isoelectric focusing

Polyacrylamide gels were stained with the sialidase substrate 2′-(4-methylumbelliferyl)-α- d- N-acetylneuraminic acid showing the activity of Vibrio cholerae and Clostridium sordellii sialidases in the gels after electrophoresis. With this fluorogenic method minimum sialidase activities of 5 μU could be determined. The sensitivity of this staining is about 10,000-fold higher compared to protein-staining with Coomassie brilliant blue. For the visualization of other proteins than sialidases the specific sialidase staining could be followed by a protein-staining method in the same gel.

Unidentified compound specifically accumulated in soybean nodules formed with H2-uptake negativerhizobium japonicumstrains

A large quantity of an unidentified compound was detected by a fluorogenic method in the soybean nodules formed with H2-uptake negative Rhizobium japonicum strains. The unidentified compound (compound X) reacted with o-phthalaldehyde in the presence of 2-mercaptoethanol giving rise to a fluorescent compound, and was not hydrolyzed in strong acid and strong base. Therefore, the compound X was considered to be one of amines and amino acids. The content of the compound X in the nodules formed with H2-uptake positive strains was remarkably lower than that in the nodules formed with H2-uptake negative strains. It was suggested that the compound X may be involved in the process of hydrogen metabolism in soybean nodules. The compound X was not detected in leaves, stems, petioles, and roots of soybean plants, but detected in H2-uptake negative strains of free-living R. japonicum. In nodules, a great portion of the compound X was distributed into cytosol fraction.

A fluorogenic method for the demonstration of human serum 5′-nucleotide phosphodiesterase isozymes after polyacrylamide gel electrophoresis

A rapid fluorogenic method for the demonstration of 5′-nucleotide phosphodiesterase in human serum has been developed. This method uses the substrate 4-methylumbelliferyl 5′-thymidylate impregnated in agarose gels or filter paper strips. Zymograms are developed in less than 30 min at 25°C, and the sensitivity of this method has been compared with that of the indigogenic method.

An investigation of flavones as fluorogenic spray reagents for organic compounds on a cellulose matrix : Part II. Detection of pesticides

Several classes of pesticides such as carbamates, s-triazines, organophosphates and chlorinated hydrocarbons have been tested. Yellow flourescent spots were observed on cellulose layers sprayed with fisetin. The visual detection limits obtained for these compounds with this new fluorogenic method range between 0.01 to 0.1 μg. The method was also extended to herbicides and fungicides of a variety of chemical structures and conclusions were drawn as to the type of fluorescence phenomenon observed. Some functional groups such as nitro and possibly amino, and molecules with a quinoid type of structure were found to quench the fluoroscence of the spray reagent; while others, such as carboxylic, cyano and methoxy groups, do not.